JPH10137569A - Gel mixing method and column shaker used therefor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a gel mixing method and a column shaker used in the method by which a gel is mixed without discharging it from a column or agitating it and stabilized to make the gel uniform and free of cracks. SOLUTION: A column shaker 1 is provided with a turntable 3 carrying a column 10 filled with a gel 11 and a motor 2 for driving the turntable 3 and shaking the column 2. When the gel 1 made nonuniform or cracked, the column 10 is shaken by the shaker to mix the gel 11 in the column 10, and the gel is made uniform and free of cracks rapidly and conveniently.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for mixing gel and a column shaking apparatus used in the method.
The present invention relates to a gel mixing method for mixing a gel filled in a column by shaking a column used for protein purification and the like, and a column shaking apparatus used in the method.

[0002]

2. Description of the Related Art Conventionally, a technique called column chromatography has been used for protein purification and the like. Column chromatography is a technique for separating and purifying a target substance using a column. In general, a method is used in which a gel serving as a stationary phase is packed in a column, and a solution (mobile phase) containing the target substance is passed through the column. Is adopted. Then, the target substance is separated and purified by utilizing the difference in distribution of the solute between the stationary phase and the mobile phase. In other words, in this technique, the target substance is separated from impurities as other substances by the difference in the degree of adsorption to the gel as the stationary phase.

[0003] For example, in the purification of interferon-α using column chromatography, an antibody against the target substance (ie, an anti-interferon-α antibody) is used to adsorb the target substance, interferon-α, to a gel.
The column is packed with a gel having immobilized on the surface. And
A solution containing interferon-α is passed through the column, and interferon-α is separated and purified by utilizing the specificity between antigen and antibody. A method of separating and purifying a target substance using a pair of substances that have a specific interaction, such as an antigen and an antibody, as in this example, is particularly called affinity chromatography. Various other methods are known as column chromatography, such as gel filtration chromatography and ion exchange chromatography.

By the way, in column chromatography, the gel may be unbalanced or cracked while passing through the column (for example, FIG. 3 (b) shows a state in which the gel is cracked). Such deviations and cracks are caused by physical factors such as pressure fluctuations and imbalanced flow when passing through the column,
Alternatively, it is thought to be caused by a change in pH or ionic strength due to a difference in liquid quality passing through the column.

[0005] As described above, when the gel is unbalanced or cracked while passing through the column, it is necessary to repair the unbalanced or crack by mixing the gel. Conventional gel mixing methods include a method in which the gel is taken out of the column and refilled, and a method in which the upper part of the column is opened and the gel is agitated from the opening using a special instrument.

[0006]

However, the above-mentioned conventional method for mixing gel has the following problems. (1) In the conventional method, the work is complicated, and extraordinary work is greatly spent. In particular, in the operation of taking out and refilling the gel, complicated operations such as attaching and detaching the column from the mounting position and taking out and refilling the gel are required. (2) As a result of the complicated work as described above, the production loss caused by stopping the separation / purification process accompanying the work is large. (3) In any of the conventional methods, there is a high risk of damaging the sterility of the gel during operation. (4) In the operation of stirring the gel, the gel may be damaged depending on the special equipment.

SUMMARY OF THE INVENTION The present invention has been made in view of the above problems, and has as its object to mix a gel without removing the gel from a column or stirring, and to stabilize the gel in a state free from unevenness and cracks. It is an object of the present invention to provide a method and a column shaker used for the method.

[0008]

According to a first aspect of the present invention, there is provided a method for mixing a gel, which comprises applying external power to a column filled with a gel and shaking the column. Is characterized by mixing the gel in the column.

According to the above-mentioned method, an external power is applied to the column, and the column is shaken to generate an inertial force in the gel in the column to mix the gel. Here, "shake" refers to all of the movement of the column that can create an inertial force in the gel, and "power" refers to the power required for the column to perform such movement. The term “column” refers to a container used for column chromatography and having a substantially cylindrical shape for accommodating a gel, and the size, capacity, material, and the like are not particularly limited.
"Gel" is a stationary phase packed in the column,
Gel particles used for gel filtration chromatography,
Used for affinity chromatography, enzymes,
It is meant to include a support bound to an antigen, antibody, hormone, inhibitor or the like.

[0010] By mixing the gel as described above, deviations and cracks generated in the gel can be quickly and easily repaired.
That is, it is not necessary to take out the gel from the column and refill the gel or directly agitate the gel with an instrument or the like. Therefore, workability is improved, and work efficiency is improved. In addition, since it is not necessary to refill the gel or to agitate the column after opening it, there is no risk of impairing the sterility of the gel, and the sterility of the gel can be maintained.

In the step of separating and purifying the target substance using column chromatography, when the gel is unbalanced or cracked, the gel is quickly and simply mixed as described above, and the gel is unbalanced during the process. Therefore, the separation of the target substance can be stabilized and the recovery rate of the target substance can be stabilized. That is, the above method achieves good homeostasis of separation and recovery of the target substance.

[0012] The method of mixing a gel according to the invention of claim 2 is as follows.
In order to solve the above-mentioned problem, the method according to claim 1 is characterized in that a power is applied so as to cause the column to reciprocate or reciprocate with rotation, and the column is shaken.

According to the above method, the gel is mixed more efficiently. In particular, it was confirmed from the experimental results described later that by applying power so as to cause the column to reciprocate with rotation, good gel mixing can be performed in a short time.
The reciprocating motion involving rotation means a reciprocating motion in which the trajectory of the column draws an arc, and the center of rotation may be at any position. For example, the reciprocating motion may be such that the column rotates about its own central axis, or the reciprocating motion may be eccentric so that the central axis of the column and the rotational axis of the motion are different.

According to a third aspect of the present invention, there is provided a column shaking apparatus comprising: a mounting means for mounting a gel-filled column; and a mixing means for mixing the gel in the column. Driving means for driving the placing means to shake the column.

According to the above configuration, when the gel in the column is unbalanced or cracked or when necessary, the mounting means is driven to shake the column filled with the gel. Thereby, an inertial force is generated in the gel in the column, and the gel is mixed.

[0016] By mixing the gel as described above, it is possible to quickly and easily repair the deviations and cracks generated in the gel.
That is, it is not necessary to take out the gel from the column and refill the gel or directly agitate the gel with an instrument or the like. Therefore, workability is improved, and work efficiency is improved. In addition, since it is not necessary to refill the gel or to agitate the column after opening it, there is no risk of impairing the sterility of the gel, and the sterility of the gel can be maintained.

In the step of separating and purifying the target substance using column chromatography, if the gel is unbalanced or cracked, the gel is mixed quickly and easily as described above, and the gel is unbalanced during the process. Therefore, the separation of the target substance can be stabilized and the recovery rate of the target substance can be stabilized. That is, the above-described apparatus achieves good homeostasis of separation and recovery of the target substance.

The meaning of each term in this claim is the same as that in claim 1.

According to a fourth aspect of the present invention, there is provided a column shaking apparatus according to the third aspect, wherein the driving means causes the column to reciprocate or reciprocate with rotation. In this manner, the above-mentioned placement means is driven.

According to the above configuration, the gel is more efficiently mixed. In particular, it was confirmed from the experimental results described later that the gel can be mixed in a short time by driving the mounting means so as to cause the column to reciprocate with rotation.

The reciprocating motion with rotation means a reciprocating motion in which the trajectory of the column draws an arc. To make the column perform such a reciprocating motion, for example, the mounting means is driven as follows. Good. That is, the column mounting surface (part) 1
A mounting means is provided so as to rotate about one axis as a rotation axis,
The driving means transmits rotational driving force to the rotating shaft so that the rotating shaft is continuously reciprocated within a predetermined angle. The column may be reciprocated after the column is mounted so that the center axis of the column coincides with the rotation axis, or the column may be reciprocated after the column is mounted so that the center axis and the rotation axis are different. You may let. However, when a large-capacity column is placed shifted from the rotation axis, high rigidity of the apparatus is required. Therefore, it is preferable to increase the rigidity of the apparatus.

In addition to the above arrangement, it is preferable to provide a distance adjusting means for adjusting the reciprocating movement distance (one-way distance) of the column. For example, if the mounting means is configured as described above and the driving means is an electric motor, and the length of a rod or arm provided between the output shaft of the motor and the rotating shaft of the mounting means can be adjusted, By adjusting the length, the exercise distance can be easily adjusted. It is also preferable to provide a speed adjusting means for adjusting the reciprocating motion speed of the column. For example, when the driving means is an electric motor, the movement speed can be easily adjusted by changing the frequency of the current supplied to the motor using an inverter. Thus, by adopting a configuration in which the movement distance and the movement speed can be easily adjusted, there is an effect that even if the size and capacity of the column or the type of the gel are changed, the mixing conditions can be easily adjusted to the optimum.

Further, it is preferable that an operation setting timer is provided in addition to the above-described configuration, and the above-described reciprocating motion is performed in the column for a predetermined time set by the timer. It is experimentally or empirically found how long the column can reciprocate under the predetermined conditions for the same column and gel, and the time required for gel mixing can be determined by a timer. The gel can be more easily and reliably mixed by setting the column and reciprocating the column for that time.

[0024]

DESCRIPTION OF THE PREFERRED EMBODIMENTS One embodiment of the present invention will be described below with reference to FIGS.

FIG. 1 shows a column shaking apparatus 1 according to this embodiment.
FIG. 2 is a side view schematically showing the external appearance of FIG.
It is sectional drawing which followed the -A line.

The column shaking apparatus 1 includes a motor 2 and a turntable 3. The turntable 3 is a mounting means for mounting and fixing the column 10 filled with the gel 11 on the mounting surface, and the motor 2 is provided in the column 10 when the gel 11 is unbalanced or cracked. Is a driving means for driving the turntable 3 to shake the column 10 in order to mix the gel 11.

The motor 2 is an electric motor with a cyclo reducer for rotating its output shaft 2a by electric power supplied from a power source (not shown). The frequency of the motor 2 is varied by using an inverter, and the rotation speed of the output shaft 2a is adjusted. Can be controlled. The inverter is controlled by the control panel 9. Therefore, by controlling how much the frequency is changed by the inverter via the control panel 9, the rotation speed of the motor 2 can be easily changed and adjusted.

The turntable 3 has a projection 3b disposed on the lower surface of a circumferential portion thereof slidably supported by a groove, and is fixed to a rotating shaft 3a at a central portion. It has a rotatable configuration. An arm 4 is attached to the rotating shaft 3a.
It is locked to one end of a rod 6 via a pin 7. On the other hand, an arm 5 is attached to the output shaft 2 a of the motor 2, and the arm 5 is locked to the other end of the rod 6 via a pin 8.

That is, the arm 4 and the arm 5 are connected by the rod 6, and the rotation of the output shaft 2a of the motor 2 causes the turntable 3 to rotate as shown in FIG. Reciprocatingly rotates around the rotation axis 3a within the predetermined angle θ determined by That is, when the motor 2 makes one rotation, the turntable 3 is rotationally driven so as to make one reciprocation within the predetermined rotation angle θ. Thereby, the column 10 on the turntable 3
Also perform reciprocating motion with rotation, and the column 10
An inertial force is generated in the gel 11 inside, and the gel 11 is mixed, so that the bias and cracks generated in the gel 11 can be quickly and easily repaired.

The length of the rod 6 can be changed and adjusted according to a known mode. By changing the length of the rod 6, the rotation angle θ of the turntable 3 is changed. In other words, by changing the length of the rod 6, the one-way distance d of the reciprocating motion of the turntable 3 and the column 10 placed thereon is changed.
Therefore, by adjusting the length of the rod 6, the one-way distance d of the reciprocating movement of the column 10 can be easily adjusted according to the size of the column 10, the type of the gel 11, and the like.

Further, as described above, the frequency of the current can be varied by using the inverter to control the rotation speed of the output shaft 2a. Therefore, the movement speed of the column 10 can be adjusted according to the size of the column 10 and the type of the gel 11. Can be easily changed and adjusted.

In addition, if an operation setting timer is provided and the column 10 is reciprocated for a predetermined time set by the timer, the mixing of the gel 11 can be performed more easily and reliably. That is, the same column 1
It is experimentally or empirically found how long the column 10 can reciprocate under predetermined conditions with respect to the gel 0 and the gel 11 so that the gel 11 can be mixed. And set the time column 1
What is necessary is just to make a reciprocating motion to zero. Thus, it is possible to avoid an undesired situation such as restarting the purification step by column chromatography without sufficiently mixing the gel 11.

When the gel 11 is mixed by the above method, the column 10 may be placed so that the center axis of the column 10 coincides with the rotation axis 3a, and then reciprocated.
The column 10 may be placed so that the center axis of the column 10 is different from the rotation axis 3a and then reciprocated. However,
When the large-capacity column 10 is placed shifted from the rotation shaft 3a, it is necessary to increase the rigidity of the column shaking device 1.

The above-mentioned column 10 is a column used for column chromatography used for protein purification and the like, and is a column-shaped container in which a gel 11, which is a stationary phase, is filled. By increasing the rigidity of the column shaking apparatus 1 as described above, the above-described mixing method of the gel 11 can be performed using the column shaking apparatus 1 even for a column 10 having a large capacity. Gel 11 filled in column 10
May be gel particles used for gel filtration chromatography, or may be a support bonded to an antibody or the like used for affinity chromatography.

Next, interferon-α (hereinafter referred to as “I
An example in which the column shaking apparatus 1 is applied to a column and a gel used in a column chromatography step which is one step for purifying FN) will be described.

After the column 10 filled with the gel 11 having the anti-IFN antibody is placed and fixed on the turntable 3, a column chromatography step is performed. First, I
The solution containing FN is passed through the column 10 and the IFN is adsorbed on the gel 11.

Next, in order to remove impurities such as foreign proteins other than IFN adsorbed on the gel 11 from the column 10, a buffer solution having a predetermined concentration is passed through the column 10 and washed. After washing, a buffer solution having a predetermined concentration is passed through the column 10 to elute and collect IFN adsorbed on the gel 11.

However, if the gel 11 is unbalanced or cracked during the above process, there is a possibility that the separation of IFN from impurities may be insufficient or the recovery rate of IFN may be reduced.

That is, in the step of passing the solution containing IFN through the column 10, if the gel 11 has a bias or a crack, the flow of the solution is bypassed, and the adsorption rate of IFN to the gel 11 decreases.

In the step of washing with a buffer solution, when the gel 11 is unbalanced or cracked, impurities such as heterologous proteins cannot be sufficiently removed, and the purity of IFN decreases.

Further, in the step of eluting IFN with a buffer solution, if the gel 11 has a bias or crack,
The recovery rate of N decreases.

In this embodiment, when the gel 11 is deviated or cracked during each step, an external force is applied to the column 10 by using the column shaking device 1 to rotate the column 10 at a predetermined rotation angle. Reciprocate rotation within θ. Thereby, column 1
Inertia force is generated in the gel 11 within 0, the gel 11 is mixed, and the bias and crack of the gel 11 are repaired.

For example, cracks 15 of the gel 11 as shown in FIG. 3 (b) were generated during the passage of the liquid shown in FIG. 3 (a), that is, during one of the steps of passing through the column 10. In such a case, first, the flow is stopped. Next, the output shaft 2 a of the motor 2 is rotated, and the driving force is transmitted to the turntable 3 via the rod 6. Thereby, the turntable 3
As shown in FIG. 3 (c), the column 10 placed on the top performs a reciprocating motion with rotation. At this time, column 1
The inertia force acts on the gel 11 filled in the space 0 and the gel 11 is mixed, so that the crack 15 generated in the gel 11 is repaired. After the restoration, as shown in FIG.
The flow can be resumed.

Next, Table 1 shows the results of experiments in which the frequency was varied by an inverter using the column shaking apparatus 1 and the gels 11 were mixed under different moving speed conditions.
The rotation angle θ of the column 10 was set to 60 degrees. Further, the distance between the surface of the gel 11 and the upper mesh of the column 10 is set to 2
0 mm, and the surface of the gel 11 was covered with seal water. The column 10 is placed at the center of the turntable 3 and the column 10 is placed eccentrically by 50 mm from the center.
The experiment was performed on the street.

[0045]

[Table 1]

In the "Evaluation" column in the rightmost column of Table 1, "X" indicates that the gel mixing effect was not obtained, and therefore, the bias or crack was not repaired, and "X" indicates that the gel mixing effect was low. The symbol “△” indicates that the gel mixing effect was large, and the mark or the crack that had been repaired was marked with “○”. In addition, the method of confirming the gel mixing effect this time is based on visual observation by a skilled person.

According to the experimental results, the eccentric width of 0 mm is also 50.
It was confirmed that the gels of both mm type could mix the gel in a short time. Also, it was recognized that the eccentric width of 50 mm had a relatively larger effect of gel mixing. The gel could be mixed more efficiently as the exercise speed was increased by controlling the inverter, that is, as the number of reciprocations (swings) per unit time was increased.

As described above, in the column shaking apparatus 1,
When the gel 11 in the column 10 is deviated or cracked, or when necessary, the turntable 3 is driven to drive the gel 11
The gel 11 is mixed by shaking the column 10 filled with. Thereby, the gel 11 is removed from the column 10.
Unevenness and cracks in the gel 11 can be quickly and easily repaired without taking out and refilling the gel 11 or directly stirring the gel 11 with an instrument or the like. At the same time, since it is not necessary to refill the gel 11 or to stir the column 10 after opening it,
The sterility of the gel 11 can be maintained.

In the step of purifying IFN, for example, using column chromatography, if the gel 11 is unbalanced or cracked, the gel is mixed quickly and easily using the column shaking apparatus 1 as described above. However, the gel 11 can be stabilized during the process so as not to be biased or cracked, so that good homeostasis of IFN separation and recovery can be achieved.

Further, in the column shaking apparatus 1, the movement distance can be changed by adjusting the length of the rod 6, and the movement speed can be easily adjusted by changing the rotation speed of the motor 2 by the inverter. Sa, volume and gel 1
Even if one type changes, it can be adjusted to the optimum mixing conditions.

By the way, in the column shaking apparatus 1, as described above, the driving force is transmitted from the motor 2 to the turntable 3 so as to cause the column 10 to reciprocate with rotation, and the column 10 is shaken. The following experiment was further performed to confirm whether the gel can be mixed by other shaking motions, that is, to confirm the difference in the gel mixing state due to the difference in column shaking motion.

The column shaking motion is shown in FIG.
Each of the four patterns A to D shown in (d) was performed a plurality of times. Pattern A is a reciprocating motion involving rotation, and was performed for three rotation angles of 30 degrees, 60 degrees, and 90 degrees. Pattern B is a linear reciprocating motion, and the linear distance is 10 cm 50 cm 100 cm 3
Followed on the street. The pattern C is a so-called figure-eight movement, and was performed in two cases, that is, a case of moving on two circles having a diameter of 50 cm and a case of moving on two circles having a diameter of 100 cm. Pattern D is a figure 8 movement with a corner, a movement on a diagonal line of 30 * 45 cm and a movement of 5
The exercise was performed in two ways, that is, exercise on a diagonal line of 0 * 75 cm.

For each experiment, a 350 mm diameter acrylic column packed with about 15 liters of gel was used. Then, without using the column shaking device 1, a column shaking motion was performed manually to confirm whether the gel could be mixed. In addition, since the column shaking motion is performed manually, the floor was marked to minimize errors in the experimental results, and the column shaking motion was performed along the marking line.

In each experiment, the column shaking exercise time was 1 minute, the time from the start of the exercise to the floating of the gel was measured, and after completion of the exercise, approximately what percentage of the gel was mixed was examined. The effect of gel mixing was evaluated as described above. Tables 2 to 5 show the experimental results of the column shaking movements of the patterns A to D, respectively.

[0055]

[Table 2]

[0056]

[Table 3]

[0057]

[Table 4]

[0058]

[Table 5]

From the above results, it was confirmed that the effect of gel mixing was greatest when the column shaking motion was a reciprocating motion involving rotation. In contrast, in the case of figure eight movement, the effect of gel mixing was hardly recognized. Even in the case of linear reciprocating movement, the effect of gel mixing was not so much recognized,
By increasing the shaking speed and mechanically shaking the column, there is a possibility that the gel can be mixed to such an extent that the bias and the crack can be repaired even in the case of the linear reciprocating motion. For example, by providing a slide table in place of the turntable 3 in the column shaking apparatus 1 and further by linearly reciprocating the column 10 in a horizontal direction by a combination of an arm and a rod, an inertia force is generated in the gel. It is possible to mix the gel to such an extent that the bias and crack can be repaired. Similarly, it can be considered that the gel can be mixed to such an extent that the bias and the crack can be repaired even if the column 10 is configured to reciprocate linearly in the vertical direction.

[0060]

As described above, the gel mixing method according to the first aspect of the present invention mixes the gel in the column by applying external power to the gel-filled column and shaking the column. How to

As a result, there is an effect that the bias and the crack generated in the gel can be quickly and easily repaired. That is, since it is not necessary to take out the gel from the column and refill the gel or directly agitate the gel with an instrument or the like, the workability is improved and the work efficiency is improved. In addition, there is no possibility that the sterility of the gel is impaired, and the sterility of the gel can be maintained.

In the step of separating and purifying the target substance using column chromatography, when the gel is unbalanced or cracked, the gel is mixed quickly and easily as described above, and the gel is unbalanced during the process. And a stable state without separation and recovery of the target substance can be achieved.

The mixing method of the gel according to the second aspect of the present invention
As described above, in the method of claim 1, a power is applied so as to cause the column to reciprocate or reciprocate with rotation, and the column is shaken.

As a result, the gel can be mixed more efficiently. In particular, by applying power so as to cause the column to reciprocate with rotation, good gel mixing can be achieved in a short time.

As described above, the column shaking apparatus according to the third aspect of the present invention comprises the mounting means for mounting the column filled with the gel and the mounting means for mixing the gel in the column. Driving means for driving the column to shake it.

As a result, there is an effect that bias and cracks generated in the gel can be quickly and easily repaired. That is, since it is not necessary to take out the gel from the column and refill the gel or directly agitate the gel with an instrument or the like, the workability is improved and the work efficiency is improved. In addition, there is no possibility that the sterility of the gel is impaired, and the sterility of the gel can be maintained.

In the step of separating and purifying the target substance using column chromatography, if the gel is unbalanced or cracked, the gel is mixed quickly and simply as described above, and the gel is unbalanced during the process. And a stable state without separation and recovery of the target substance can be achieved.

According to a fourth aspect of the present invention, in the column shaking apparatus according to the third aspect of the present invention, in the configuration of the third aspect, the driving means is configured to cause the column to reciprocate or reciprocate with rotation. This is a configuration for driving the placing means.

Thus, the gel can be mixed more efficiently. Particularly, by driving the mounting means so as to cause the column to reciprocate with rotation, good mixing of the gel can be achieved in a short time.

[Brief description of the drawings]

FIG. 1 is a side view schematically showing an appearance of a column shaking apparatus according to an embodiment of the present invention.

FIG. 2 is a sectional view taken along line AA of FIG.

FIG. 3 is an explanatory diagram showing a procedure for repairing a crack in a gel in a column when the crack occurs.

FIG. 4 is an explanatory diagram showing each motion pattern of a column shaking motion performed in an experiment.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Column shaking apparatus 2 Motor (drive means) 3 Turntable (placement means) 4.5 arm 6 Rod 7.8 pin 9 Control panel 10 Column 11 Gel

 ──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Seiji Ito 5-1 Sokai-cho, Niihama-shi, Ehime Prefecture Sumitomo Pharmaceutical Co., Ltd. (72) Inventor Akinori Okada 3-139, Nitta-cho, Niihama-shi, Ehime Sumitomo Inside Chemical Engineering Co., Ltd. (72) Inventor Yoshihisa Ishikawa 3-39, Nittacho, Niihama-shi, Ehime Prefecture Inside Sumitomo Chemical Engineering Co., Ltd.

Claims (4)

[Claims] 1. A method for mixing gels, wherein external power is applied to a column filled with gel and the column is shaken to mix the gel in the column. 2. The gel mixing method according to claim 1, wherein a power is applied so as to cause the column to reciprocate or reciprocate with rotation, and the column is shaken. 3. A mounting means for mounting a column filled with gel, and a driving means for driving the mounting means and shaking the column to mix the gel in the column. Characteristic column shaker. 4. A column shaking apparatus according to claim 3, wherein said driving means drives said placing means so as to cause said column to reciprocate or reciprocate with rotation.